(r4) Barrick Lab :: QPCRToQuantifyPlasmidCopyNumber

---+ Absolute QPCR for quantification of plasmid copy number in _E. coli_

This protocol is based on methods described in Lee _et al_ (2006), [[http://www.sciencedirect.com/science/article/pii/S0168165605007509][link to paper]].

---++ Designing primers for QPCR

You can design your primers manually, or alternatively, we use this [[https://www.idtdna.com/Primerquest/Home/Index][online tool from IDT]].

---++ Preparation of DNA sample templates for QPCR

   1 Grow an overnight culture of each strain harboring your plasmid of interest in LB media supplemented with antibiotic 
      * You may want to start 3-5 replicate cultures for each strain
   1 Dilute your saturated cultures 1:100 into fresh media and let grow for 2-3 hours until the cells reach a mid-exponential phase (OD<sub>600</sub> = ~0.4-0.6)
   1 For each sample, pellet 1 ml of cells for 5 minutes at 3,000 RPM
   1 Extract total DNA from these pellets using a genomic DNA isolation kit 
      * Our lab uses [[https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-extraction/genomic-dna-extraction/purelink-genomic-dna.html][this one]]

---++ Preparation of DNA for plasmid and standard curves

   1 For the gDNA standard curve, you will need to extract total DNA from wild-type _E. coli_ strain not containing any plasmids 
      * It is generally good practice to use the same _E. coli_ strain harboring your plasmid of interest
   1 For the plasmid standard curve, you will need to mini-prep your plasmid

---++ QPCR using SYBR Green I dye, Part 1: Setting up a plate

   1 Set up standard curves for your gDNA and plasmid samples, these will also help you calculate your primer efficiencies (should be between 0.8-1.1) 
      * To generate standard curves, dilute your gDNA and plasmid templates in ten-fold increments
      * A total of 7 dilutions is enough to make a good standard curve, your CT values should be between 5-30
   1 Normalize your sample templates to 2ng/µL 
      * Make sure to dilute these so the concentrations fall within the range of the standard curves
   1 Once your DNA templates have all been diluted, you can being to set up a 96- or 384- well plate to run your qPCR experiment 
      * For 96-well plates the reaction volumes are as follows:

%TABLE{dataalign="center"}%
| *Template* | *10µM F primer* | *10µM R primer* | *SYBR Green PCR Mix* | *ddH<sub>2</sub>O* |
| X | 0.75 | 0.75 | 7.5 | template - X |

   1 Run your qPCR plate using the following cycling conditions: 
      * Step 1 = Hold Stage 
         1 50°C - 02:00
         1 95°C - 10:00
      * Step 2 = PCR Stage 
         1 95°C - 00:15
         1 54°C - 01:00
         1 Go to step 2-1, 40X
      * Step 3 = Melt Curve 
         1 95°C - 00:15
         1 54°C - 01:00
         1 95°C - 00:15

---++ QPCR using SYBR Green I dye, Part 2: Analyzing your data

   1 Calculate the primer efficiencies by plotting CT vs DNA concentration for the gDNA and plasmid standards, and using [[https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/qpcr-efficiency-calculator.html][this website]]
   1 If your efficiencies are between 0.8-1.1, then calculate the ratio of plasmid:gDNA for each of your samples

-- Main.DaciaLeon - 15 Dec 2016
Barrick Lab > ProtocolList > QPCRToQuantifyPlasmidCopyNumber
Contributors to this topic
DaciaLeon, SimonDAlton, DanielDeatherage
Topic revision: r4 - 2017-02-23 - 20:32:53 - Main.DaciaLeon